This is an ongoing comprehensive program concerned with study of the structure, composition, function, regulation, and interaction of the enzyme complexes of the mitochondrial electron transport/oxidative phosphorylation system, which were previously isolated, purified and characterized by the applicant and coworkers. Those under study in recent years have been Complex I (NADH:ubiquinone oxidoreductase), Complex II (succinate:ubiquinone oxidoreductase), Complex III (ubiquinol:cytochrome c-oxidoreductase), and Complex V (ATP synthase). Studies planned for the next grant period include the following: Complex I: Structure of iron-sulfur flavoprotein (FP) and iron-sulfur protein (IP) fragments; study of electron transfer from FP to IP; isolation of iron-sulfur (FeS)-containing polypeptides of the hydrophobic protein fraction; delineation of the inhibition sites of dicyclohexylcarbodiimide and ethoxy-formic anhydride (EFA); attempt to differentiate the Em of FeS clusters N3 and N4; study possible effect of guanidine on site 1 coupling. Complex II: Separation of two subunits of cytochrome b560; study of role of each subunit (and of b560 heme) in electron transfer from succinate dehydrogenase (SDH) to ubiquinone (Q) and in reconstitution with Complex III; attempt at resolution of SDH into its two subunits without altering their FeS cluster structure for localization of clusters S1, S2 and S3. Complex III: Delineate inhibition sites of EFA, myxothiazol, stigmalellin, antimycin, etc.; test the individual steps of the Q-cycle hypothesis; study effect of various inhibitors on Complex III structure. Complex V: Isolate subunits 6 and 8 by electroelution; use antibodies to subunits 6 and 8 to study their possible roles in ATP synthesis and hydrolysis; check whether there is a polypeptide in mitochondria and Complex V that is structurally related to subunit-6; localize the F0 thiols whose modifications result in (a) uncoupling, and (b) inhibition of ATPase activity. Mechanism of Oxidative Phosphorylation: Continuation of studies on the mechanism of energy transfer in ATP synthase (we have evidence that, in the absence of proton translocation, modifications at specific F0 loci result in changes in the mode of ATP binding to F1); continuation of studies on the kinetic modalities of ATP synthesis and the role of electrochemical potential of Proton in interconversion of these kinetic modes; extension of these studies to E. coli membranes, chloroplasts and chromatophores.